Name: Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids
Uploaded: 2023-10-13T12:30:00
Description: Watch this Scientific Journal Video about Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids at JoVE.com

setting up an acoustic assembly system and cell culture harvesting for generating uniform cell spheroids

एक 3 डी ध्वनिक विधानसभा डिवाइस का उपयोग वर्दी सेल गोलाकार की स्केलेबल पीढ़ी

3D in vitro culture models, which provide more in vivo-like structural and morphological characteristics compared to conventional 2D culture models, have been recognized as promising systems in various biomedical applications such as tissue engineering, disease modeling, and drug screening1,2,3. As one type of 3D culture model, cell spheroids typically refer to cell aggregation, creating 3D spheroidal structures characterized by enhanced cell-cell and cell-matrix interactions4,5,6. Therefore, fabricating cell spheroids has become a powerful tool for enabling diverse biological studies.
Various techniques, including hanging drop7, non-adhesive plates8, or microwell devices9, have been developed to obtain spheroids. In principle, these methods commonly facilitate cell assembly by utilizing physical forces such as gravitational force while minimizing interactions between cells and the substrate. However, they often involve labor-intensive processes, have low productivity, and pose challenges for controlling spheroid size10,11. Importantly, the production of spheroids with the desired size and uniformity in sufficient quantity is of utmost importance to satisfy specific biological applications. In contrast to the above-mentioned methods, acoustic waves, as one type of external-force-driven technique12,13,14, have shown potential for mass manufacturing of cell spheroids with high quality and throughput, based on the principle of enhancing cell aggregation through external forces15,16,17,18. Unlike electromagnetic or magnetic forces, acoustic-based cell manipulation techniques are non-invasive and label-free, enabling spheroid formation with excellent biocompatibility19,20.
Commonly, standing surface acoustic waves (SAWs) and bulk acoustic waves (BAWs)-based devices have been developed to generate spheroids, utilizing the acoustic nodes (ANs) produced by corresponding standing acoustic fields21,22,23. Particularly, acoustic assembly devices based on BAWs, with the merits of convenient manufacture, easy operation, and excellent scalability, have gained attention for fabricating cell spheroids24,25. We have recently developed a facile BAWs-based acoustic assembly device with the ability to generate spheroids with high throughput26. The proposed device consists of a square polymethyl methacrylate (PMMA) chamber with three lead zirconate titanate (PZT) transducers arranged respectively in the X/Y/Z plane. This arrangement enables the creation of a 3D dot-array pattern of levitated acoustic nodes (LANs) for driving cell assembly. Compared to previously reported BAWs- or SAWs-based devices, which can only create a 1D or 2D array of ANs27,28,29, the present device enables a 3D dot-array of LANs for rapid cell aggregate formation within the gelatin methacryloyl (GelMA) solution. Subsequently, cell aggregates matured into spheroids with high viability within the photocured GelMA scaffolds after three days of cultivation. Finally, a large number of spheroids with uniform size were easily obtained from the GelMA scaffolds for downstream applications.

लेखक स्पॉटलाइट: उच्च गुणवत्ता वाले 3 डी सेल स्फेरॉइड संस्कृति के लिए एक मचान मुक्त ध्वनिक विधानसभा विधि का विकास 

सेल स्फेरॉइड के बड़े पैमाने पर विनिर्माण के लिए तीन आयामी ध्वनिक विधानसभा उपकरण

3D in vitro culture models, like cell spheroids, have been widely used in the fields of tissue engineering, disease modeling, and drug screening. Our research focuses on developing and invest per engineering method to fabricate spheroids or other 3D in vitro culture models with high quality for diverse biological studies. In the current protocol, the spheroids grew within the GelMA scaffold and are retrieved at the final layback, dissolving the scaffold, causing additional procedures.We will try to acoustically assemble cell aggregates in a cultural medium to enable them to grow within spheroids without scaffold. Compared to magnetic assembly techniques, acoustic waves can directly assemble cells in a label-free manner without potential toxic effects on cells. Besides, our protocol enables cell spheroids to be assembled in a three-dimensional array, substantially increasing throughput.

Watch this Scientific Journal Video about Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids at JoVE.com

Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids  

यूनिफ़ॉर्म सेल स्फेरॉइड पैदा करने के लिए एक ध्वनिक असेंबली सिस्टम और सेल कल्चर हार्वेस्टिंग की स्थापना

शुरू करने के लिए, ध्वनिक उपकरण के कक्ष को पांच मिनट के लिए 75% अल्कोहल के साथ बाँझ करें। फिर, बाँझ पीबीएस के साथ डिवाइस को साफ करें और इसे एक घंटे के लिए यूवी प्रकाश से विकिरणित करें। कक्ष के इंटीरियर के शीर्ष-दृश्य अवलोकन के लिए एक खुर्दबीन चरण पर बाँझ ध्वनिक उपकरण माउंट करें।डिवाइस के किनारे पर एक डिजिटल माइक्रोस्कोप रखें जो पीजेडटी ट्रांसड्यूसर से मुक्त है, जिससे कक्ष के इंटीरियर का साइड-व्यू अवलोकन सक्षम हो सके। स्वतंत्र रूप से तीन PZT ट्रांसड्यूसर से श्रृंखला में तारों को तीन पावर एम्पलीफायरों और फ़ंक्शन जनरेटर के तीन आउटपुट चैनलों से कनेक्ट करें। प्रत्येक पीसीटी ट्रांसड्यूसर के लिए फ़ंक्शन जनरेटर पर सेटिंग्स को प्रोग्राम करें, साइनसोइडल तरंगों, आवृत्ति और आयाम जैसे मापदंडों को निर्दिष्ट करें।डीएमईएम में संस्कृति 3 सीए कोशिकाओं को टी 25 सेल संस्कृति फ्लास्क में 10% एफबीएस और 1% पेनिसिलिन स्ट्रेप्टोमाइसिन के साथ पूरक किया जाता है। जब कोशिकाओं 80% confluent हो जाते हैं, पीबीएस के साथ दो बार संस्कृति धोने. पीबीएस को हटाने के बाद, फ्लास्क में 0.05% ट्रिप्सिन-ईडीटीए के दो मिलीलीटर जोड़ें और सेल डिटेचमेंट के लिए 37 डिग्री सेल्सियस पर इनक्यूबेट करें।ट्रिप्सिनाइजेशन को रोकने के लिए फ्लास्क में पूर्ण सेल संस्कृति माध्यम के दो मिलीलीटर जोड़ें। सेल निलंबन को 15-मिलीलीटर ट्यूब में स्थानांतरित करें और पांच मिनट के लिए 200 ग्राम पर अपकेंद्रित्र करें।

Research

JoVE Journal

Bioengineering

Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...